Cell surface-associated proteins which bind native type IV collagen or gelatin.

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY-D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine-containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N-glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin-sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.

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Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and ?2?1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Experimental Cell Research ◽

10.1016/0014-4827(91)90395-b ◽

1991 ◽

Vol 195 (2) ◽

pp. 443-457 ◽

Cited By ~ 23

Author(s):

I SCHAFER

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Bullous Pemphigoid ◽

Basal Cell ◽

Type Iv Collagen ◽

Human Keratinocytes ◽

Heparan Sulfate Proteoglycan ◽

Sulfate Proteoglycan ◽

Collagen Gels ◽

Type Iv

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Cell adhesion to extracellular matrix regulates the life cycle of integrins.

Molecular Biology of the Cell ◽

10.1091/mbc.6.12.1781 ◽

1995 ◽

Vol 6 (12) ◽

pp. 1781-1791 ◽

Cited By ~ 48

Author(s):

S L Dalton ◽

E Scharf ◽

R Briesewitz ◽

E E Marcantonio ◽

R K Assoian

Keyword(s):

Extracellular Matrix ◽

Life Cycle ◽

Cell Surface ◽

Matrix Protein ◽

Type Iv Collagen ◽

Surface Expression ◽

Extracellular Matrix Protein ◽

Integrin Subunit ◽

Type Iv ◽

Beta 1 Integrin

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.

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CD44/chondroitin sulfate proteoglycan and alpha 2 beta 1 integrin mediate human melanoma cell migration on type IV collagen and invasion of basement membranes.

Molecular Biology of the Cell ◽

10.1091/mbc.7.3.383 ◽

1996 ◽

Vol 7 (3) ◽

pp. 383-396 ◽

Cited By ~ 96

Author(s):

J R Knutson ◽

J Iida ◽

G B Fields ◽

J B McCarthy

Keyword(s):

Cell Migration ◽

Cell Adhesion ◽

Cell Surface ◽

Tumor Cell ◽

Melanoma Cell ◽

Cell Invasion ◽

Type Iv Collagen ◽

Human Melanoma ◽

Type Iv ◽

Beta 1 Integrin

Tumor cell invasion of basement membranes (BM) represents one of the critical steps in the metastatic process. Tumor cell recognition of individual BM matrix components may involve individual cell adhesion receptors, such as integrins or cell surface proteoglycans, or may involve a coordinate action of both types of receptors. In this study, we have focused on the identification of a cell surface CD44/chondroitin sulfate proteoglycan (CSPG) and alpha 2 beta 1 integrin on human melanoma cells that are both directly involved in the in vitro invasion of reconstituted BM via a type IV collagen-dependent mechanism. Interfering with cell surface expression of human melanoma CSPG with either p-nitro-phenyl-beta-D-xylopyranoside treatment or anti-CD44 monoclonal antibody (mAb) preincubation (mAb) preincubation inhibits melanoma cell invasion through reconstituted BM. These treatments also strongly inhibit melanoma cell migration on type IV collagen, however, they are ineffective at inhibiting cell adhesion to type IV collagen. Purified melanoma cell surface CD44/CSPG, or purified chondroitin sulfate, bind to type IV collagen affinity columns, consistent with a role for CD44/CSPG-type IV collagen interactions in mediating tumor cell invasion. In contrast, melanoma cell migration on laminin (LM) does not involve CD44/CSPG, nor does CD44/CSPG bind to LM, suggesting that CD44/CSPG-type IV collagen interactions are specific in nature. Additionally, anti-alpha 2 and anti-beta 1 integrin mAbs are capable of blocking melanoma cell invasion of reconstituted BM. Both of these anti-integrin mAbs inhibit melanoma cell adhesion and migration on type IV collagen, whereas only anti-beta 1 mAb inhibits cell adhesion to LM. Collectively, these results indicate that melanoma cell adhesion to type IV collagen is an important consideration in invasion of reconstituted BM in vitro, and suggest that CD44/CSPG and alpha 2 beta 1 integrin may collaborate to promote human melanoma cell adhesion, migration, and invasion in vivo.

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The use of 1nm silver enhanced gold probes for the detection of skin antigens

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162077 ◽

1990 ◽

Vol 48 (3) ◽

pp. 902-903

Author(s):

J.P Cassella ◽

H. Shimizu ◽

A. Ishida-Yamamoto ◽

R.A.J. Eady

Keyword(s):

Type Iv Collagen ◽

Freeze Substitution ◽

Collagen Type Iv ◽

Electron Microscopic ◽

Normal Human Skin ◽

Type Iv ◽

Type Vii Collagen ◽

Keratin Filaments ◽

Normal Human ◽

Phosphate Buffered Saline

1nm colloidal gold with silver enhancement has been used in conjunction with a low-temperature post-embedding (post-E) technique for the demonstration of skin antigens at both the light microscopic (LM) and electron microscopic (EM) levels.Keratin filaments and basement membrane zone (BMZ) associated antigens in normal human skin (NHS) were immunolabelled using antibodies against keratin 14, 10, and 1, the carboxy-terminus and collagenous portion of type VII collagen, type IV collagen and bullous pemphigoid antigen (BP-Ag).Fresh samples of NHS were cryoprotected in 15% glycerol, cryofixed in propane at -190°C, subjected to freeze substitution in methanol at -80°C and embedded in Lowicryl K11M at -60°C. Polymerisation of the resin was initiated under UVR at - 60°C for 48 hours and continued at room temperature for a further 48 hours. Semith in sections were air dried onto slides coated with 3-aminopropyltriethoxysilane. The following immunolabelling protocol was adopted: Primary antibody was applied for 2 hours at 37°C or overnight at 4°C. Following washing in Dulbecco’s phosphate buffered saline (PBSA) a biotinylated secondary antibody was applied for 2 hours at 37°C. The sections were further washed in PBSA and 1nm gold avidin was applied. Sections were finally washed in PBSA and silver enhanced.

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